Artificial cultivation of the hog-cholera virus



canny ll blll l...

rnnnnnrcx rnonsonnn, or sronx crrr, town.

ARTIFIGIAh CULTIVATION F THEE] HOG-GHUIJERA Villtlfh.

instincts.

lilo llrawtn i To all whom it may concern lle itlrnoivn that l, lnnonnrcn Pnonscnrnn, a citizen of the. limited States, residing att'lioui; City, in the countyot "Woodbury and State oi llou'a, haveinvented an Artificial lllultivation ot the Hog-tholera Virus Suit ablei'or Prevention and Cure of Hog Cholera, or which the following is afull, clear,

and exact description.

lily inrentionhas relation to the artificial cultivation ot thehogcholera virus from the natural virus by nieans of new culture mediawhich it have discovered. lhese culture media may also be used for themelting ct subcultures,

" vantages. llhis result has not, to my knowledge, been previouslyattained for various reasons. In the first place, the hog-choleramicroorganism on account of its extremely small size, belongs totheclass of filterable viruses. andhas heretofore been regarded asinvisible. Furthermore, these micro-organ isrns. are very'd lficult togrow outside the living" organisms in which they naturally grow, andthey can be made" to grow artionly in" specially prepared culture media,J j i l have discovered thatf it is possible to render such organismsvisible by special staini ig nietliodsgf and havefalso invented culture,med a in WlllCll such organisms can be successfully cultivated, both incultures and in subcultures.

primary The present invention has indie particular reference to thegeneral methodjofcultrvation of the artificlal virus, theparticularculture media and the methods of preparing such culture media,and other novel features as herein set forth, and as clalmed.

The hog cholera.micro-organism is a'"minutecoc'cus on the limit ofInicroscopialvisihili y, being about one-tenth R micron Sin diarneter,It occurs either as single coccus,

mg l in Pair'sjbr asshort chains or congl'onierated cocci. I havediscovered that the microorganism can be made visible Specification ofLetters Patent.

in the primary lesions by staining With methylene azure or methyleneviolet, or in general, with staining solutions containing anilin dyesbelonging to the thiazzin grouo.

ts a rule, the YHUS is most excellently stained with the free base oimethylene azure.

The rirus can be cultivated 'l'roni the blood and urine or the organjuices lroin all the lnfected organs. l or practical purposes thecultivation of the rirus trorn the blood. or filtered organ juice is themost suitable method. The virulence oi the artificially cultivated viruscan be attenuated by means of vvell lrnoivn physical and chemicalagencies, such as heating to a certain degree, the addition ofchemicals, such as carbolic acid, etc. These methods are Well known inthe preparation of bacterial vaccine, and are intended tor use in theproduction oil a hop," cholera vaccin.

lC-he blood of pigs sullering from natural hog-cholera infection isusually containinated with secondary invaders such as hue, Suiscpticus,Eu'ipcsttfcr and other members oil the paratyphoid and enteritis group,and which will interfere With the cultivation of the virus. It is,therefore, necessary to re move the secondary invaders by filtration andto infect healthy susceptible pigs With filtered virus. For thefiltration a dense porcelain filter may be used. lEnperieuce has shownthat the blood of pigs from the fifth to seventh day of the disease isusually i L sufficiently free 0t secondary invaders and contains thehog-cholera virus in a sufliciently purestate, to use after filtration,for

deathaseptically. Theblood is preferably jkept fluid With sodiumcitrate, using 80 jcubiccentimeterscf a solution to 1000 cubiccentimeters oi blood. Instead of using blood so obtained, filtered serumor hemolyzed blood maybe used, and also the filtered juice from theorgans oi infected pigs, such as heart, lungs, kidneys, spleen, liverand lymph glands.

The term virus as used herein and in the claims Will be understood toapply to anyor all of these/substances,

As above stated, the hog-cholera ,virus Wlll not grow lncommon culturemedia. In

"thepracticeoilny discovery, I have ob- Pn-tented llllar. filfl,ilhfillli Application filed January 2?, 1919. Serial No. fl'lfihth.

served that the best growth is observed in compound culture media madefrom pigs organs and blood with the addition of other suitable culturemedia prepared from the tissue of other animals such as cattle, goats,rabbits, fowls, sheep, horses and so forth. Human and animal transudatesand exudates may be used. For continuous growths 011 these media theaddition of hemoglobin and serum and fresh or auto-claved tissue,preferably spleen, is essential. It is also advisable that one at least,of these ingredients be derived from pigs, the virus thereby growingbetter.

For the cultivation of the virus, culture media composed of a solutionof completely hydrolyzed protein containing amino-acids and polypeptidesand containing hemolyzed blood, (dissolved hemoglobin) can also beemployed.

The following culture media, together with methods of preparing the sameare typical examples.

Culture M ediu/ N0. 1.

(1.) Hog serum is diluted 1 to 5 or 1 to 8 with distilled water andautoclaved at ten pounds pressure for thirty minutes.

(2.) 500 grams lean minced beef meat are mixed with 1000 cubiccentimeters distilled water and autoclaved for one hour at ten poundspressure, filtered and 1% peptone added to the filtrate. The eptonebroth or bouillon is autoclaved for fteen minutes at ten pounds pressureand filtered again.

(3.) 500 grams fresh minced hog kidneys mixed with 1000 cubiccentimeters water is autoclaved for one hour then filtered and 1%peptone is added to the filtrate. The mixture is then autoclaved againfor fifteen minutes and again filtered.

(4.) The three ingredients thus prepared are then mixed in the followingproportions:

1000 cubic centimeters hog serum water,

1000 cubic centimeters beef bouillon,

400 cubic centimeters kidney bouillon,

.3% dextrose.

(5.) Add normal sodium and potassium hydroxid solution to such an extentthat 10 cubic centimeters of the mixture will require preferably forexact neutralization, not more than two-tenths cubic centimeters of aone-tenth normal hydrochloric acid solution, using phenol red asindicator.

Oultwre Medium N o. 2.

(1.) Hog serum water made as described above.

(2.) Beef bouillon made as described above.

(3.) Kidney bouillon made as described above.

(4.) The mucous membrane of several cleansed and washed hog stomachs isremoved, ground finely and treated as follows:

500 grams minced mucous membrane,

1000 cubic centimeters distilled water,

10 cubic centimeters hydrochloric acid (chemically pure).

This mixture is kept at a temperature of 50 degrees C., for twenty-fourhours; is then filtered through absorbent cotton then sterilized in theautoclave for fifteen minutes at ten pounds pressure. The mixture isthen filtered and neutralized at room temperature with a normal sodiumand potas- Culture M cdz'um N 0. 3.

(1.). An equal volume of hog stomach digest, after it has been kept for24 hours and 50 (1, and filtered through cotton, is mixed with an equalvolume of hog serum, and is digested again for 21 hours, at 50 C. At theend of the 24 hours, the mixture is autoclaved for 30 minutes at 10pounds pressure. If the digesting is carried out properly a littlesediment will be formed by the autoclaving. The hot mixture is thenfiltered through absorbent cotton or filter paper and neutralized with amixture of normal sodium and potassium hydroxid solution, using phenolred as an indicator. After neutralizing, the mixture is again autoclavedfor 15 minutes at 10 pounds pressure and filtered again.

(2.) To 1000 cubic centimeters of digested hog serum and beef bouillon,mixed equally, are added 200 cubic centimeters kidney bouillon and .3%dextrose. The beef and kidney bouillon are made as described underCulture Medium No. 1. The mixture is adj usted to the same alkalinity asdescribed under Culture Medium No. 1.

M airing the culture.

Either one of the three above described culture media may be filled intest tubes or flasks, and to each test tube or flask a piece of freshpig spleen is added before sterilizing. About 3% of spleen is 'asuitable amount. Sterilize three times in streaming steam, for 15minutes, for three consecutive days.

tered through asbestos WOOl until clear, then through a coarse porcelainfilter and finally through a dense porcelain filter. Before adding; thefiltered hemolyzed blood it should he tested for sterility.

The culture media alcove described may he used for making cultures fromnatural virus. drawn blood or filtered organ ymce of a pip,"

' infected with hog-cholera is added to these culture media in theproportion of from. a. to 8 cubic centimeters of blood to'lll cubiccentimeters of the culture media. The tulies are lrept at approuimately8'1 degrees tern. perature for from 3 to 8 days. if grovvth taltes placea difi'use slight cloudiness ob served. For microscopical examination. afew drops of culture are taken and smears made direct; or better, theculture is diluted With sterile Water in proportion l:l(l,'is

' centrifuged, and smears are made of the sediment. The smears arestained with Gram solution and counter-stained. with concern tratedcarbolfuchsin for one-half minute. The micro-organisms usually occur inclumps as very minute Gram negative cocci.

The artificial virus may be ,used for the production of antidmg'choleraserum or for the active immunization (in the form of attenuated vaccin)or for the simultaneous inununiuation for pigs and'hogs.

instead ofusing; the 'vvhole culture, the virus may he centrifugedtherefrom and the sediment taken up With sodium chlorid s0ltl-' tion ofabout 35% and the emulsified sediment used for injection.

llt will be understood that the culture media described above aretypical only and that other culture media, possessing similarcharacteristics and brought to the proper ion-concentration, may heemployed. .Tt will also be understood that the methods of malt" ing theculture media which I have described and the proportions given are those"Which I have found to give good, results. I do not desire to limitmyself thereto as many modifications and variations will readily occurto bacteriologists skilled in this art.

The ter1nspig and hog are used interchangeably throughout thisspecification and the claims thereof.

ll claim:

l. The herein described method for the artificial cultivation of thehog'cholera virus which consists in obtaining a virus-con taining'material from infected pi s and free from secondary invaders, andadding" such also With a bouillon made from lridney ti sue, adjustingthe miuture to a very on For this purpose the aseptically thereto,adding spleen thereto and stem mg, then adding sterile hemolyced blood,-

virus-containing'd material to a culture medium prepared from animaltissue, and adjusted" to an ion-concentration approaching neutrality andthereafter maintaining the culture medium under conditions favorable forthe development of the micro-organ ism.

2. The herein described method for-the artificial cultivation of thehog-cholera virus, Which consists in matting an animal serum Water,mining the same'ivith a bouillon made from the; tissue of animals anddegree of alkalinity, adding carhohydra and mixing the sterile culturemedium vvith the virus, and thereafter maintaining the culture mediumunder conditions favorable for the development of the micro-organ ism.

3. The herein described method for the artificial cultivation of thehog-cholera 'Vl-c rus, Which consists in melting a hog serum Water,mining the same With a bouillon made from tissue of animal other thanhog, and also With the'houillon made from the hog kidney and digestedhog stomach, adjusting the mixture to an ion-concentration appreachingneutrality, adding dextrose, plac ing the medium in a container, adding;pig s spleen, sterilizing, adding hemolyzed hlood, and mining thesterile culture medium With the viru and incubating until themicroorganisms are suiliciently developed.

d. The herein described method for the artificial cultivation of thehog-cholera virus which consists in digesting hog serum, min ing thesame from a bouillon made from. the tissure of animal other than hog"and also with a bouillon made from hog kidney, adjustingthe mintur'eto avery slight degree of allralinity,adding dextrose, placing the medium ina container, adding pig spleen, sterilizing, then adding hemolyzedblood, and mixing the sterile culture medium With the virus, andincubating until the micro organisms are sufiiciently developed.

5. The method of cultivating the hog cholera virus, which consists ingrowing the same in a medium consisting of a bouillon made from animaltissue and adjusting" to a very small degree of alkalinity, said mediumcontaining also spleen and hemolyzed blood to promote continuous growthin the suhculture.

6. The method of cultivating hog-cholera virus Which consists in growingthe same in y l a medium containing a bouillon made from animal tissueand containing spleen and hemolyzeol blood, at least one of said ingradients being derived from pi 7. A. culture medium for the artificialcultivation of hog-cholera virus comprising a lilll ltlli lltl llti

tivation of hog-cholera virus, comprising a mixture of hog serum Water,a bouillon made from the tissue of animals other than hogs, and abouillon made from hog kidneys, together with a carbohydrate.

10. A culture medium for the artificial cultivation of hog-choleravirus, comprising a mixture of hog serum water, a bouillon made fromanimal tissue and a bouillon made from kidneys, together with acarbohydrate and pig spleen.

11. A culture medium for the artificial cultivation of hog-choleravirus, comprising a mixture of 110g serum water, a bouillon made fromthe tissue of animals other than hogs, and a bouillon made from hogkidneys, together with dextrose, pig spleen and hemolyzed blood.

12. A culture medium for the artificial cultivation of hog-cholera viruscomprising amixture of 110g serum water, a bouillon made from the tissueof animals other than hog, and a bouillon made from hog kidneys anddigested hog stomachs.

13. A culture medium for the artificial cultivation of hog-choleraserum, comprising a mixture of hog serum water, a bouillon made from thetissue of animals other than hogs, and a bouillon made from hog kidneysand digested hog stomachs, together with dextrose and hog spleen.

14. A culture medium for the artificial cultivation of hog-choleravirus, comprising a mixture of hog serum water, a bouillon made from thetissue ofanimals other than hogs, and a bouillon made from hog kidneysand digested hog stomachs, together with dextrose and hog spleen andhemolyzed blood.

15. A culture medium for ,the artificial cultivation of hog-choleravirus, prepared from animal tissue and adjusted to proper alkalinity,and also containing pig spleen and hemolyzed pig blood,

l6. A culture medium for the artificial cultivation of hog-choleravirus, prepared from animal tissue other than hog tissue, and containinghog spleen and hemolyzed hog blood.

17 A culture medium suitable for the artificial cultivation ofhog-cholera virus, comprising a mixture of animal tissue brothscontaining a dissolved hemoglobin.

18. In the production of artificial hogcholera virus the steps ofremoving all secondary organisms from a juice from an infected hog,inoculating therewith a culture medium containing hemoglobin, andmaintaining the culture medium under conditions proper for thedevelopment of the virus.

19. As a new product, an artificial pure culture of hog cholera virus,in a nutrient medium containing dissolved hemoglobin.

20. As a new product, an attenuated artificial pure culture of hogcholera virus, in a nutlrient medium containing dissolved hemo- 10 in.

b In testimony whereof I have hereunto set my hand. 1

FREDERICK PROESCHER.

